Project summary: Variola virus (smallpox) is regarded to be 1 of the most significant threats as a bioterrorism agent. Because smallpox was eradicated as a human disease before the molecular tools were available to dissect host immune responses, little is known about the molecular, cellular, and organismal characteristics of host immunity to poxvirus infection. The urgent scientific need for these studies is emphasized by a recent National Academy of Science project on smallpox, which stated that "it is important to define the innate immune response to a poxvirus infection, including ... cellular receptors involved in activation of elements of the innate immune system". Recent studies suggest that Toll-like receptors (TLR) may be 1 of the key cellular determinants of innate immunity to poxviruses. TLR are crucial for the host innate immune system to recognize and respond to invading pathogens, and TLR signaling also regulates the adaptive immune response. The objective of this research is to develop whole animal imaging techniques to study poxvirus pathogenesis in mouse models, enabling real-time studies of TLR effects on viral replication and host response in the context of a living animal. These imaging technologies will be used to accomplish these specific aims: 1) determine the extent to which TLR signaling through 2 different adaptor proteins (MyD88 and Trif) limits replication and systemic spread of vaccinia virus, the established model for poxvirus infection; and 2) establish effects of TLR signaling on distribution and trafficking of T lymphocytes to sites of vaccinia infection. This research will provide a detailed understanding of TLR in poxvirus pathogenesis, providing an essential foundation for developing strategies to control smallpox infection and its spread. Furthermore, our studies will establish innovative imaging approaches for studying poxvirus pathogenesis in living mice. These imaging strategies can be applied to other aspects of poxvirus- host pathogenesis and pre-clinical testing of new vaccination strategies or anti-viral compounds. Relevance: This research will establish functions of a specific family of molecules that likely are important in the immune response to vaccinia virus, the virus used as a model of smallpox. A key component of the application is validating imaging techniques to detect and analyze the virus and defined host immune cells in a living animal. Collectively, the research is expected to facilitate the development of improved vaccines and treatments for smallpox, thereby enhancing our ability to prevent the use of smallpox for bioterrorism. [unreadable] [unreadable] [unreadable]